Project Summary My career goals are to obtain a tenure-track faculty position and to establish a laboratory that uses quantitative approaches to elucidate complex in vivo immune regulation. The objective of my work is to understand how genetic or environment-induced variation in negative regulators can influence the sensitivity of innate immune cells to inflammatory stimuli. Research at the interface of immunology and computation promises to advance our understanding of dynamic signaling circuits that translate stimuli quality and quantity into the appropriate functional response, thus informing therapeutic strategies that target these pathways. My previous experience in assessment of in vivo immune cell activation, quantitative signaling analysis, and computational modeling puts me in an excellent position to work at the intersection of these fields. Innate immune sensing of microbial stimuli must be tightly regulated to support robust protective inflammatory responses to infection, while avoiding inflammation upon minor challenges at barrier sites. The threshold for inflammatory responses is dictated by strict control of MAPK activity. While the activating components of this pathway have been well studied, a fundamental challenge in inflammation research is to understand the negative regulation that scales these signals to facilitate quantitative decision-making within cells. Genome wide association studies have linked MAPK-regulating phosphatases with chronic inflammatory diseases of the respiratory and gastrointestinal tracts, and changes in microbiota composition in these barrier tissues are also associated with inflammatory disease. Considering the mixed success of attempts to therapeutically target MAPK in a variety of such diseases, elucidating the influence of disease-associated genetic factors and microbiota-dependent stimuli on MAPK regulation may inform treatment optimization. The objective of this proposal is to illuminate regulatory mechanisms that support quantitative control of microbe-induced signaling thresholds in macrophages and to determine whether these thresholds are distinctly regulated in barrier tissues. The studies proposed in Aim 1 will use a combination of quantitative experimental and computational modeling approaches to address the role of phosphatase regulation at the transcriptional and post-translational levels on scaling of MAPK activation dynamics. These efforts will yield insight into how changes in the expression or activity of key regulators, resulting from genetic variation, tissue-specific signals, or pharmacological manipulation, can tune macrophage sensitivity to microbial products. The experiments proposed in Aim 2 will interrogate distinct signaling tuning in barrier tissues, specifically the small intestine and lung, and will address the role of microbiota-dependent stimuli in regulation of macrophage signaling, both in the steady state and in response to minor inflammatory challenge. Tuning of macrophage signaling may play a critical role in dampening inflammatory responses in barrier tissues, and thus our efforts to elucidate regulation and dysregulation of this process will inform mechanistic links between disease-associated genetic factors, changes in the microbiota, and inflammatory disease development. While pursuing the research strategy described above, I will work with Drs. Martin Meier-Schellershiem, Michel Tremblay, and Yasmine Belkaid to gain technical skills and enhance my expertise in computational modeling, protein phosphatases, and study of the microbiota. My current mentor, Dr. Ronald Germain, has supported me in forming these collaborations and in presenting my research prominently at several international scientific meetings. These opportunities have helped me to establish my reputation as an investigator at the intersection of quantitative biology, signaling, and innate immunity transitioning to independence, and to form long-term colleagues to provide support and outside evaluation of my work during this transition. I will also seek advice from my early career mentors Drs. Peter Savage and Suzanne Gaudet, who have recently navigated the process of career transition. By supporting the completion of the proposed aims and the associated career development opportunities, this award will help me to establish the necessary foundation for additional funding and for my successful transition to scientific independence.